JBUON 2013; 18(3): 739-745
ISSN: 1107-0625, online ISSN: 2241-6293 • www.jbuon.com
E-mail: [email protected]
ORIGINAL ARTICLE
The role of flow cytometry in the diagnosis of nonHodgkin’s lymphoma, Hodgkin’s lymphoma, granulomatous
inflammation and reactive lymph node specimens
E. Gunduz1, M. Celebioglu2, O. Meltem Akay1, H. Uskudar Teke1, F. Sahin Mutlu3,
Z. Gulbas4
1
Eskisehir Osmangazi University School of Medicine, Department of Hematology, Eskisehir; 2Medline Hospital, Clinic of Internal
Medicine, Eskisehir; 3Eskisehir Osmangazi University School of Medicine, Department of Biostatistics, Eskisehir; 4Anadolu Health
Center, Bone Marrow Transplantation Center, Kocaeli, Turkey
Summary
Purpose: In this study we aimed to compare the flow cytometry (FC) results of patients with B cell lymphoma, T
cell lymphoma, Hodgkin’s lymphoma, granulomatous inflammation and reactive lymph node and investigate the
role of FC in malignant or non malignant conditions.
Methods: Ninety patients were divided into 5 groups according to histopathology results. Patients were compared
according to cytokeratin and positivity percentage of the following surface markers: CD45, CD19, CD5, CD19-CD5, CD4,
CD8, CD3,CD16-CD56, CD10, CD10-CD19, CD23, CD20,
CD4-CD8, CD3-CD16-56, CD30, CD38, kappa and lambda
light chains, CD20-CD23. Patients were also compared according to the intensity of the expression (exp) of same markers. ROC curve analysis was performed for CD19+ cell percentage, CD38 exp, kappa/lambda and lambda/kappa ratios.
Introduction
Lymphadenopathy can be reactive or secondary to infections, lymphomas, leukemias, metastases from carcinoma, autoimmune diseases,
reactions to drugs etc. [1]. Traditionally, the technique of choice for diagnosis of lymphadenopathy was based on the histopathologic study of
paraffin-embedded tissue. Currently, the use of
immunohistochemistry (IC) for detecting cell spesific antigens is essential in classifying tumors,
identifying prognostic factors and identifying targets for therapy [2]. However, IC in routine clinical practice has limitations: analysis subjectivity,
Results: 1) Kappa/lambda and lambda/kappa ratios can
distinguish B cell lymphoma from T cell lymphoma, Hodgkin’s lymphoma, granulomatous inflammation and reactive lymph node; 2) CD19+ cell percentage can distinguish T
cell lymphoma from Hodgkin’s lymphoma, granulomatous
inflammation and reactive lymph node; 3) CD38 exp can
partly distinguish B cell lymphoma from T cell lymphoma,
Hodgkin’s lymphoma, granulomatous inflammation and
reactive lymph node and T cell lymphoma from granulomatous inflammation, T cell lymphoma from reactive lymph
node, Hodgkin’s lymphoma from reactive lymph node.
Conclusion: Flow cytometry has a role in distinguishing
lymphomas from non malignant lesions.
Key words: flow cytometry, granulomatous inflammation, kappa/lambda ratio, lymphoma, reactive lymph node
limited reproducibility and time investment [3,4].
FC immunophenotyping requires only a small
sample. It is able to detect aberrant cells at a frequency of 1/10000 cells [5]. The large number of
specific monoclonal antibodies and the possibility
of combining 4 or more fluorochromes to precisely define the cell profile and the neoplastic nature
of lymphoid proliferations make FC a more sensitive and quicker tool than is IC [6-9]. Evaluating
several antigens on 1 cell, including cytoplasmic
antigens, gives quantitative results and can detect
small abnormal cell populations against a reactive background. It can also assess coexpression
of antigens on subsets of cells and the relative
Correspondence to: Eren Gunduz, MD. Eskisehir Osmangazi University School of Medicine, Department of Hematology, 26480
Eskisehir, Turkey. Tel: +90 222 2398466, Fax: +90 222 2393774, E-mail:[email protected]
Received: 21/12/2012; Accepted: 12/01/2013
740
Flow cytometry in the diagnosis of lymphomas and nonlymphoma conditions
density of surface antigens. It has additional advantage of being able to identify a small population of abnormal cells that may not be apparent
on morphology alone [10,11].
Although Fromm et al. [12] reported that 8 or
more color FC can diagnose classical Hodgkin’s
lymphoma in lymph nodes with high sensitivity
and specifity, the usefulness of FC in contributing
to the diagnosis of classical Hodgkin’s lymphoma involving lymph nodes has been limited [13].
There is limited data about FC in granulomatous
inflammation and reactive lymph nodes.
The aim of this retrospective study was to report our experience in assessing the contribution
of FC immunophenotyping in the diagnosis of B
cell lymphoma, T cell lymphoma, Hodgkin’s lymphoma, granulomatous inflammation and reactive
lymph nodes.
Methods
We retrospectively analysed the medical records
of 90 consecutive patients who had persistent enlargement of lymph nodes and underwent biopsy for both
FC and histopathology analysis. Patients were divided
into 5 groups according to histopathology diagnosis:
B cell lymphoma (N=18;Group 1), T cell lymphoma
(N=14;Group 2), Hodgkin’s lymphoma (N=20;Group 3),
granulomatous inflammation (N=15;Group 4) and reactive lymph node (N=13;Group 5).
Lymph node specimens were obtained in the operation room with intact capsule and sent complete to
laboratory in RPMI culture medium. Lymph node specimen without fat and necrosis in 10 mm3 volume was
placed in 50 mm medicon with DAKO medimachine
system. 0.5-1.5 ml suspension buffer (RPMI or Phosphate buffered saline/PBS were added. Medicons were
placed in medimachine and tissue was cut into pieces
with microknives during 2-2.5 min. Medicon was taken out of medimachine and the suspension was filtered
through 70 µm filcon. Cells were washed with PBS for
2-3 times and resuspended with cell pellet. A cell concentration between 5-40,000 was adjusted.
Monoclonal antibody panel included CD45 FITC
(Fluorescein isothiocyanate) / CD14 PE (Phycoerythrin), Mouse IgG2a FITC / Mouse IgG1 PE, CD20 FITC /
CD5 PE, CD4 FITC / CD8 PE, CD3 FITC / CD56 PE, CD3
FITC / HLA-DR PE, CD10 FITC / CD19 PE, CD23 FITC /
CD19 PE, kappa FITC / CD19 PE, lambda FITC / CD19
PE, CD30 PE, cytokeratin FITC and CD38 PE (Becton
Dickinson Bidscienes, CA, USA).
Test procedure
Thirteen 12x75 mm tubes were used. Patients’
name and the antibodies used were written on the
tubes. Ten µl vortexed monoclonal antibody was put in
JBUON 2013; 18(3): 740
each tube. A hundred µl of cell suspension was added,
vortexed and incubated in the dark at room temperature for 20 min. Two ml of red blood cell lysing were
added and rested at room temperature for 10 min, then
centrifuged at 1800 rpm for 5 min. and the supernatant
was removed. This washing process was repeated once
more. Five hundred µl PBS were added into the tubes
and acquisition of data was made. Cell group was gated and positive antigens were analyzed. Mononuclear
cells were separated for kappa and lambda and washed
twice with PBS.
Patients were compared according to cytokeratin
and positivity (+) percentage of the following surface markers: CD45, CD19, CD5, CD19-CD5, CD4, CD8,
CD3,CD16-56, CD10, CD10-CD19, CD23, CD20, CD4CD8, CD3-CD16-56, CD30, CD38, kappa, lambda, CD20CD23. Patients were also compared according to the
intensity of the expression (exp) of the following antigens: CD45, CD19, CD5, CD4, CD4-CD8, CD8, CD8-CD4,
CD3, CD16-CD56, kappa, lambda, CD10, CD10-CD19,
CD 19-CD10, CD20, CD23, CD20-CD23, CD23-CD20,
CD30, CD38.
Statistics
Analysis of variance ( Tamhane, Tucey), and Least
Significant Difference (LSD), Kruskal-Wallis and x2
tests were used to analyze the results. The sensitivity and specifity of positivity of surface markers and
cytokeratin for the diagnosis were evaluated with the
positive (PPV) and negative predictive value (NPV). A
receiver-operating characteristic (ROC) curve was used
to determine the diagnostic accuracy and cut-off value.
Data were expressed as median or mean ± standard deviation (SD). A p-value<0.05 was considered as statistically significant.
Results
After FC analysis of lymph node specimens,
cytokeratin and CD45+, CD19+,CD5+, CD19+CD5+, CD4+, CD8+, CD3+, CD16,56+, CD10+,
CD10+-CD19+, CD23+, CD20+, CD4+-CD8+,
CD3+-CD16,56+, CD30+, CD38+,kappa+, lambda
+, CD20+-CD23+ cell percentages of each group
were compared (Table 1). There was no statistically significant difference between groups according to CD19+-CD5+, CD16-56+, CD10+-CD19+,
CD4+-CD8+, CD3+-CD16-CD56+, CD30+, CD38+,
CD20+-CD23+ cell percentages. Groups were also
analyzed according to the intensity of the expression of antigens (Table 2). Cytokeratin was negative in all groups.
According to ROC curve analysis obtained
for patients in each group, 11% cut-off for CD19
distinguished T cell lymphoma from Hodgkin’s
lymphoma with 87.5% sensitivity and 92.5%
Flow cytometry in the diagnosis of lymphomas and nonlymphoma conditions
741
Table 1. Comparison of positive cell percentages in lymph node for each group
CD19+
CD20+
CD19± Kappa+
CD19+ lambda+
CD5+
CD4+
CD8+
CD3+
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
18
13
16
13
11
18
13
16
13
11
28
12
19
15
13
28
12
19
15
13
28
14
20
15
13
28
14
20
15
13
28
14
20
15
13
28
14
20
15
13
70 ± 30
6±5
30 ± 16
33 ± 22
36 ± 14
51 ± 33
7±6
11 ± 6
23 ± 15
23 ± 8
40 ± 39
5±3
13 ± 11
20 ± 12
18 ± 7
28 ± 40
5 ±3
12 ± 8
16 ± 9
14 ± 7
22 ± 18
58 ± 25
56 ± 18
50 ± 18
52 ± 16
19 ± 18
32 ± 28
47 ± 18
38 ± 13
43 ± 17
19 ± 18
32 ± 28
47 ± 18
38 ± 13
43 ± 17
27 ± 21
54 ± 35
64 ± 18
51 ± 26
58 ± 17
1
2
**
**
**
**
**
*
**
Kappa± lambda+
Lambda± kappa+
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
28
14
20
15
13
28
12
19
15
13
28
12
19
15
13
2.7 ± 3.1
3.8 ± 7.2
4.4 ± 4.4
3.1 ± 1.1
4 ± 2.7
18.99 ± 28.5
1.26 ± 0.6
1.03 ± 0.6
1.2 ± 0.3
1.26 ± 0.22
14.3 ± 29.6
0.9 ± 0.3
2.3 ± 3.7
0.8 ± 0.2
0.8 ± 0.1
4
5
Comparison (Probability)
2
3
4
5
**
**
**
**
*
*
**
**
**
**
**
**
**
*
**
*
**
**
**
**
1
CD4± CD8+
3
**
**
**
**
*
*
*
*
*p<0.05, **p<0.01. SD: standard deviation
JBUON 2013; 18(3): 741
742
Flow cytometry in the diagnosis of lymphomas and nonlymphoma conditions
Table 2. Comparison of positive cell percentages in lymph node for each group
Group
N
Median
1
27
193
Comparison (Probability)
1
Kappa exp
2
12
505
3
19
264
4
15
212
**
12
242
**
5
1
27
214
2
12
427
3
19
427
4
15
158
*
5
12
605
*
SD=4
21
469
2
7
636
**
3
8
892
**
4
11
274
**
7
304
**
x2=16
CD45 exp
5
*
x =10.3
1
5
4
SD=4
2
CD38 exp
3
**
x2=13.9
Lambda exp
2
SD=4
1
26
437
2
13
526
3
17
408
4
13
276
5
13
471
x =5.98
SD=4
2
*p<0.05, **p<0.01
Exp: expression, SD: standard deviation
specificity, T cell lymphoma from granulomatous
inflammation with 76.9% sensitivity and 92.3%
specifity, T cell lymphoma from reactive lymph
node with 100% sensitivity and 92.3% specificity.
ROC curve analysis was also generated for
the intensity of the expression of the above mentioned antigens. A cut-off value 291 for CD38exp
distinguished T cell lymphoma from granulomatous inflammation with 81.8% sensitivity and
100% specificity. Cut-off 304 for CD38exp distinguished T cell lymphoma from reactive lymph
node with 57.1% sensitivity and 100% specificity. Cut-off 291 for CD38exp distinguished Hodgkin’s lymphoma from granulomatous inflammation with 81.8% sensitivity and 87.5% specificity.
Cut-off 638 for CD38exp distinguished Hodgkin’s
lymphoma from reactive lymph node with 100%
sensitivity and 62.5% specificity.
Sensitivity and specifity of kappa/lambda and
lambda/kappa light chain ratios for certain tresholds in B cell lymphoma patients were also evalJBUON 2013; 18(3): 742
uated (Tables 3 and 4). When the thresholds for
kappa/lambda ratio were chosen as 1.5, 2, 2.5 and
3, the sensitivity and specificity were found highest for 3. When the threshold for lambda/kappa ratio were chosen as 1.5, 2, 2.5 and 3 the sensitivity
were found highest for 2.
Discussion
In this study we evaluated the cell distribution
in lymph nodes of patients with B cell lymphoma,
T cell lymphoma, Hodgkin’s disease, granulomatous inflammation and reactive lymph node by using FC and comparing the results for each group.
CD19+ cell percentage in B cell lymphoma patients was higher than in T cell lymphoma, Hodgkin’s lymphoma, granulomatous inflammation
and reactive lymph node. This was an expected
finding since CD19 is a well known B cell associated marker. Lower CD19+ cell percentage was
found significant in distinguishing T cell lym-
Flow cytometry in the diagnosis of lymphomas and nonlymphoma conditions
Table 3. Positive and negative predictive values of
kappa/lambda ratio in B cell lymphoma
Treshold
1.5
Sensitivity
94.1
Specifity
PPV
96.6
88.9
NPV
98.3
2
100
96.7
88.9
100
2.5
100
96.6
88.2
100
3
100
100
100
100
PPV: positive predictive value, NPV: negative predictive value
Table 4. Positive and negative predictive values of
lambda/kappa ratio in B cell lymphoma
Treshold
Sensitivity
Specifity
PPV
NPV
1.5
100
89.8
60
100
2
100
98.6
87.5
100
2.5
100
98.3
87.5
100
3
100
98.3
87.5
100
PPV: positive predictive value, NPV: negative predictive value
phoma from other disorders, but we could not
find a similar comparison in the literature. CD20+
cell percentage in B cell lymphoma patients was
found higher than in T cell lymphoma and Hodgkin’s lymphoma but was not different from granulomatous inflammation and reactive lymph node.
In our study kappa+-CD19+ cell percentage in
B cell lymphoma patients was higher than in T
cell lymphoma and Hodgkin’s lymphoma patients.
This percentage was lower than in granulomatous
inflammation and reactive lymph node in T cell
lymphoma patients. Kappa/lambda and lambda/
kappa ratios in B cell lymphoma were higher than
in T cell lymphoma, Hodgkin’s lymphoma, granulomatous inflammation and reactive lymph node.
These findings were compatible with the literature which showed that lymphomas of B cell origin can be detected by their expression of B surface markers and light chain restriction [14-17].
It is more difficult to identify phenotypically
abnormal T cells than abnormal B cells. FC plays
only a part in the diagnosis of T cell lymphomas.
The identification of populations of abnormal T
cells may be achieved by demonstrating aber-
743
rant T cell antigen expression or by identifying
restricted populations of T cells. Complete lack of
staining for one or more pan T cell antigen(s) may
prove that T cell population present is abnormal
[18]. In our study CD5+ and CD3+ cell percentages in B cell lymphoma were lower than in T cell
lymphoma, Hodgkin’s lymphoma, granulomatous inflammation and reactive lymph nodes. We
also found that CD5 and CD3 increase in similar
amounts in T cell lymphoma, Hodgkin’s lymphoma, granulomatous inflammation and reactive
lymph node and as a result can not be used in distinguishing the aforementioned diseases.
We found CD4+ cell percentage in B cell lymphoma was lower than in Hodgkin’s lymphoma,
granulomatous inflammation and reactive lymph
node. We concluded that CD4+ cell percentage
is important in distinguishing B cell lymphoma
from others but could not find similiar data in the
literature.
Another finding in our study was lower kappa
exp in B cell lymphoma from T cell lymphoma,
granulomatous inflammation and reactive lymph
node. Kappa exp in granulomatous inflamation
and reactive lymph node was lower than in T cell
lymphoma and Hodgkin’s lymphoma patients. We
could not find a report about the importance of
kappa exp in distinguishing these diseases but
our study showed that kappa exp is important in
distinguishing T cell lymphoma, granulomatous
inflammation and reactive lymph node.
Lambda exp was found lower in B cell lymphoma from T cell lymphoma and reactive lymph
node but higher than in granulomatous inflammation. Lambda exp in granulomatous inflammation was lower than in Hodgkin’s lymphoma.
Lambda exp in reactive lymph nodes was higher
than in Hodgkin’s lymphoma and granulomatous
inflammation. We concluded lambda exp helps in
distinguishing reactive lymph nodes and granulomatous inflammation.
In our study CD38 exp in B cell lymphoma
was lower than in T cell lymphoma and Hodgkin’s
lymphoma but higher than in granulomatous
inflammation and reactive lymph nodes. CD38
exp in granulomatous inflammation and reactive
lymph node was lower than in T cell lymphoma
and Hodgkin’s lymphoma. Although it was not reported before in the literature we found CD38exp
was significant in distinguishing B cell lymphoma from Hodgkin’s lymphoma and granulomatous inflammation.
When we compared the CD19+ cell percents
JBUON 2013; 18(3): 743
744
Flow cytometry in the diagnosis of lymphomas and nonlymphoma conditions
of patients with T cell lymphoma and Hodgkin’s
lymphoma the diagnosis was in favor of Hodgkin’s
lymphoma if the CD19+ cell percent was >11%
(sensitivity: 87.5% ; specificity: 92.5% ). When we
compared CD19+ cell percents of patients with T
cell lymphoma and granuloma inflammation on
the diagnosis was in favor of inflammation if the
CD19+ cell percent was >11(sensitivity: 76.9% ;
specificity: 92.3%). When we compared the CD19+
cell percents of patients with T cell lymphoma and
reactive lymph node the diagnosis was in favor
of reactive lymph nodeif the CD19+ cell percent
was >11% (sensitivity: 100% ; specificity: 92.3%).
We could not find similar data in the literature
but concluded that CD19+ cell percentage is important in distinguishing T cell lymphoma from
Hodgkin’s lymphoma, granulomatous inflammation and reactive lymph node.
In our study a cut-off <291for CD38exp was
in favor of granulomatous inflammation (sensitivity: 81.8% ; specificity: 100%). When we compared ROC curve analysis obtained for patients
with T cell lymphoma and reactive lymph node a
cut-off <304 for CD38exp was in favor of reactive
lymph node (sensitivity:57.1% ; specificity:100%).
When ROC curve analysis obtained for patients
with Hodgkin’s lymphoma and granulomatous inflammation a cut-off <291 for CD38exp was in
favor of granulomatous inflammation (sensitivity: 81.8% ; specificity: 87.5%). When we compared
ROC curve analysis obtained for patients with
Hodgkin’s lymphoma and reactive lymph node a
cut-off <638 for CD38exp was in favor of reactive
lymph node (sensitivity: 100%; specificity: 62.5%).
FC is one of the most convenient and powerful
means for establishing B cell clonality. The kappa/
lambda ratio in nonneoplastic specimens is usually in the range of 1/1 to 2/1. B cell lymphomas
express a single clonal light chain so this ratio
is generally increased or decreased. Jorgensen et
al. [5] suggest investigating the case more closely
if kappa/lambda ratio is >3/1 or if there is a significant lambda excess. Mature B cell neoplasms
usually express only one class of immunglobulin
light chain. The normal ratio of kappa/lambda is
1/1 to 2/1. Monoclonality is diagnosed when this
ratio is >4/1 or >1/2 [19].
JBUON 2013; 18(3): 744
Chizuka et al. [20] analysed the predictive value of kappa/lambda light chain ratio in 105 B cell
lymphoma patients. They found highest sensitivity (92.5%) and specificity (73.1%) when kappa/
lambda ratio was equal to 2. PPV was 90% and
NPV 72% for this ratio. Ten percent of patients
with a kappa/lambda ratio >2 did not have B cell
lymphoma and more than 20% of patients did not
have B cell lymphoma when this ratio was <2. In
the same study 38 of 53 (72%) B cell lymphoma
patients had a kappa/lambda ratio higher than
2 and kappa light chain was dominant in 22 of
them. Kappa/ lambda ratio was lower than 2 in
15 patients. Only 2 of the patients without B cell
lymphoma had kappa/lambda ratio higher than 2.
Diagnoses were chronic lymphadenitis and tuberculosis in these patients.
We also evaluated the specificity and sensitivity for certain kappa/lambda and lambda/kappa
light chain ratio thresholds in B cell lymphoma
patients. The highest specificity (100%) and sensitivity (100%) for kappa/lambda ratio was found
for threshold 3 (PPV 100%, NPV 100%). Specificity was decreased for values lower than 3. Kappa/
lambda ratio was higher than 3 in 14 of 28 (50%)
B cell lymphoma patients. Kappa light chain was
dominant in 17 of them. The highest specifity
(100%) and sensitivity (100%) for kappa/lambda
ratio in B cell lymphoma was found for threshold
2 (PPV 87.5%, NPV 100%). Lambda/kappa ratio
was higher than 2 in 6 of 28 (21%) B cell lymphoma patients and lambda light chain was dominant
in 11 of them. Kappa/lambda ratio higher than 3
or lambda/kappa ratio higher than 2 by FC analysis of lymph node suggests B cell lymphoma.
To our kowledge this is the first study in the
literature comparing the FC results of B cell lymphoma, T cell lymphoma, Hodgkin’s lymphoma,
granulomatous inflammation and reactive lymph
nodes together. Our results show that CD 19,
CD 20, CD 3, CD4, CD5+ cell percentages, kappa,
lambda, CD 38 exp and kappa/lambda, lambda/
kappa ratios are important in distinguishing between these diseases. Kappa/lambda ratio higher
than 3 or lambda/kappa ratio higher than 2 by FC
analysis of lymph node suggest B cell lymphoma.
Flow cytometry in the diagnosis of lymphomas and nonlymphoma conditions
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